1. Field of the Invention
The present invention relates generally to the detection of aldehydes in such media as aqueous solutions, blood, urine and other body fluids and more specifically, to a reagent, the method of making the reagent, and a test kit for using the reagent in the detection of aldehydes and more specifically, malondialdehydes.
2. Description of the Related Art
It is by now common knowledge that stress in mammalian subjects develops directly or indirectly into a display of oxygenated activities that quickly changes the usual reduced state of the body. This hyperoxygenated statexe2x80x94can cause great physical imbalance and actual physical damage that can change to pathological states, which in turn, develop into atherosclerotic plaques. Such plaques can result in the deposition of high lipid levels particularly in a blockage of arteries that can cause a cessation of blood flow to the heart with a resulting heart attack. This is one of but many human disease states that are caused by free radical attacks from the hyperoxygenated state caused by stress.
The oxidative stress state can be measured from the release of aldehydes, particularly as dialdehydes, from the breakdown of long-chain polyunsaturated fatty acids.
We have found that a fuchsin based calorimetric test can measure the released aldehyde in a rapid, easily performed home test kit by using the reagent described below with a small quantity of urine. The resulting color formed is compared to a calibrated test strip to assess the level of stress from a negative value through levels of +1 to +3.
Various test for aldehydes are, of course known. For example, as disclosed at page 395 in xe2x80x9cQualitative Analysis by Spot Testsxe2x80x9d, Third Edition, authored by F. Feigl and published by Elsevier Publishing Company, Ind., a drop of sample solution which may contain aldehydes in mixed with 2 ml of 72 percent sulfuric acid in a test tube. A small amount of solid chromatropic acid (1,8-dihydrooxynapthianene-3 6 disulfate) is added and the test tube is heated in a 60 degree C. water bath for about ten minutes. A bright violet color appears in the presence of aldehyde sensitivity of the test is reportedly about 3 ppm of aldehyde.
In another aldehyde test, described at pages 339-340 of the Feigl publication, a drop of aqueous (or alcoholic) solution suspected of containing an aldehyde is treated on a spot plate with a drop of sulfurous acid and a drop of fuchsin/sulfiric acid and allowed to stand. A red to blue color appears within about two to thirty minutes, according to the amount of aldehyde present in the test solution being tested. Such test is reportedly sensitive to about one microgram of formaldehyde in a drop of solution being tested. The problem with such a test, and other known aldehyde tests, is that the tests are not quickly and easily performed. The first above-described test, for example, requires heating of a test tube of solution in a constant temperature water bath for ten minutes. As a result of aldehyde tests not being quickly and easily performed, there may be a tendency for the tests for aldehydes not to be performed as frequently as they should or otherwise would be tested.
A further aldehyde test is disclosed in U.S. Pat. No. 6,165,797. As is therein disclosed, urine is tested for presence of malondialdehyde by mixing it with a reagent consisting of about 90-110 parts 20% acetic acid, about 13.5 to 16.5 parts Ingredient A and about 4.5 to 5.5 part Ingredient B, wherein Ingredient A is comprised of sodium metabisulfite, phosphoric acid and the ionized water in the proportions of about 18-22 grams sodium metabisulfite, 9-11 ml of concentrated phosphoric acid, and about 450-550 ml of dionized water, and ingredient B is comprised of basic fuchsin and Ingredient A in the proportion of about 0.45-0.55 grams basic fuchsin to about 90-110 ml Ingredient A. It has been found that the reagent thus formed is much to acidic because of the use of acetic acid and the reaction time when urine is added thereto is extremely quick and results in a color change occurring as a result of the pH of the urine. Thus, false positives occur and it becomes very difficult if not impossible to detect the presence of aldehydes in the urine on a consistent and reliable basis.
For these and other reasons, there is required an improved reagent for reliably detecting the presence of very small amounts of aldehydes in biological fluids so as to detect adverse biological oxidation and therefore decrease medical costs. It is also required that such reagent be packaged in a manner than an individual may easily use it in a home self test.
An object of the invention is to provide a simple test for determining the presence of aldehyde in a test specimen.
Another object is to provide a test reagent which produces a color change when mixed with a test specimen containing aldehyde.
According to the present invention, a testing solution or reagent for detecting the presence of aldehyde, particularly in an aqueous solution, urine and other body fluids, comprises a solution of phosphoric acid, sodium metabisulfite, basic fuchsin and deionized water. The preferred proportions of the elements for a 1 liter batch are 10 grams sodium metabisulfite, 10 ml concentrated phosphoric acid, 5 grams basic fuchsin and about 1000 ml of water.
Basic fuchsin changes color in an acidic solution, relative to the amount of aldehyde present in the specimen being tested. Basic fuchsin is a purple powder which reacts with aldehydes in the skin, urine or blood plasma. With low or no aldehydes present, you get no color development. With moderate or high levels of aldehydes you get color gradations roughly dependent on the level of aldehydes present. The amino group of the fuchsin couples with the aldehyde to produce the pink to purple color approximately dependent on the amount of aldehyde present in the biological fluids such as blood or urine. The color developed depends on the pH, which is controlled by the amount of acid present.
Sodium metabisulfite is a reducer to stop the interference of oxygen from air. Metabisulfite ties up free oxygen so that only the aldehydes react with the fuchsin group. Establishing a nitrogen blanket over the reagent mixture gives greater shelf life to the reagent by stopping any oxygen reaction with the reagent. The phosphoric acid stabilizes the pH.
Aldehydes are released from the breakdown of long chain polyunsaturated fatty acids by free radical attacks.
High levels of aldehydes are found in a variety of diseases and abnormal metabolism states such as coronary artery disease, diabetes, and Parkinson disease.
A method for testing for aldehydes in an aqueous solution, urine, blood and other body fluids comprises mixing a test specimen such as urine suspected of containing traces of aldehydes into the testing reagent described above, and observing for color formation after at least 2 minutes, preferably 2-5 minutes. The preferred proportions are about 0.9 to 1.1 ml of the test specimen to about 0.1-0.2 ml of the testing reagent. If the mixture does not become colored within such time, the test specimen contains less than about 2 ppm of aldehyde. If, however, the mixture becomes colored, for example, pinkish-purple, within the time specified, or sooner, aldehyde presence of greater than 2 ppm in the test specimen is inferred, the more intense the color, the greater the concentration of aldehyde.
Preferably, the testing reagent is contained in a sealed ampule or vial. Test specimen is introduced to the reagent in a snap-type ampule. A simple, efficient, reliable and rapid test for the presence of aldehyde in aqueous solution is thereby provided. Typically, the ampule along with an appropriate dispenser such as a syringe or pipette and a container for collecting the specimen are housed in a package bearing a color chart. This provides an easy, reliable way for an individual to test for the presence of aldehydes in his or her urine.